Method for the identification of antagonists of a phenylthiocarbamide/bitter taste receptor

ABSTRACT

The present invention relates to bitter-taste receptors and their role in bitter taste transduction. The invention also relates to assays for screening molecules that modulate, e.g. suppress or block bitter taste transduction, or enhance bitter taste response.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage Application of internationalApplication Number PCT/EP2003/010691, filed Sep. 25, 2003; which claimsthe benefit of U.S. Provisional Application Ser. No. 60/413,298, filedSep. 25, 2002, which are incorporated herein in their entirety.

BACKGROUND OF THE INVENTION

Investigators have recently turned their attention to understanding thebiological mechanisms of taste, and in particular bitter taste. For areview of the literature see, for example, Science 291, 1557-1560.(2001); Cell 100, 607-610 (2000); Neuron 25, 507-510 (2000); Nature 413,219-225. (2001); and J. Biol. Chem. 277, 1-4 (2001).

Bitter taste is aversive, and as such provides humans with a mechanismof protection against poisonous substances, which are generallybitter-tasting compounds. More subtly, bitter-tastants also affect thepalatability of food, beverages, thereby influencing human nutritionalhabits as is more fully discussed by Drewnowski in “The Science andComplexity of Bitter Taste”, Nutr. Rev. 59, 163-169 (2001). They alsoaffect the palatability of other ingestibles such as orally administeredpharmaceuticals and nutraceuticals. Understanding the mechanism ofbitter taste transduction has implications for the food andpharmaceutical industries. If the bitter taste transduction pathway canbe manipulated, it may be possible to suppress or eliminate bitter tasteto render foods more palatable and increase patient compliance in oralpharmaceutics.

Taste transduction involves the interaction of molecules, i.e., tastantswith taste receptor-expressing cells which reside in the taste budslocated in the papillae of the tongue. Taste buds relay information tothe brain on the nutrient content of food and the presence of poisons.Recent advances in biochemical and physiological studies have enabledresearchers to conclude that bitter taste transduction is mediated byso-called G-protein coupled receptors (GPCRs). GPCRs are 7 transmembranedomain cell surface proteins that amplify signals generated at a cellsurface when the receptor interacts with a ligand (a tastant) whereuponthey activate heterotrimeric G-proteins. The G-proteins are proteincomplexes that are composed of alpha and beta-gamma subunits. They areusually referred to by their alpha subunits and classified generallyinto 4 groups: G_(alpha s, i, q and 12). The G_(alpha q) type couplewith GPCRs to activate phospholipase C which leads to the increase incellular Ca²⁺. There are many G_(q)-type G-proteins that are promiscuousand can couple to GPCRs, including taste receptors, and these so-called“promiscuous” G-proteins are well known to the man skilled in the art.These G-proteins dissociate into alpha and beta-gamma subunits uponactivation, resulting in a complex cascade of cellular events thatresults in the cell producing cell messengers, such as calcium ions,that enable the cells to send a signal to the brain indicating a bitterresponse.

There is also anatomical evidence that GPCRs mediate bitter tastetransduction: clusters of these receptors are found in mammalian tastecells containing gustducin. Gustducin is a G-protein subunit that isimplicated in the perception of bitter taste in mammals, see for exampleChandrashekar, J. et al., Cell 100, 703-711 (2000); Matsunami H. et al.,Nature 404, 601-604 (2000); or Adler E. et al., Cell 100, 693-702(2000). cDNAs encoding such GPCRs have been identified, isolated, andused as templates to compare with DNA libraries using in-silicodata-mining techniques to identify other related receptors. In thismanner it has been possible to identify a family of related receptors,the so-called T2R family of receptors, that have been putativelyassigned as bitter receptors.

To-date, however, it is not clear as to whether all the bitter tastereceptors have been discovered. Further, of those that have beendiscovered, many have not been matched, or paired, with ligands, andapplicant is aware of very few published studies wherein rigorousmatching has been undertaken. Chandrashekar, J. et al. in Cell 100,703-711 (2000), has expressed a human T2R receptor, the so-called hT2R4receptor, in heterologous systems and looked at the in vitro response ofthis receptor. They found that it provided a response to the bittercompounds denatonium and 6-n-propyl-2-thiouracil. However, theconcentrations of bitter tastants needed to activate the hT2R4 receptorwere two orders of magnitude higher than the thresholds reported inhuman taste studies, and so it is not clear that the protein encoded bythe hT2R4 gene is a functional bitter receptor. The authors of theChandrashekar et al. article also looked at a number of mouse T2Rreceptors with a range of stock bitter-tasting chemicals of disparatechemical structure. However, no study has looked at receptor responsesto bitter ligands that are problematic in the food and pharmaceuticalindustries, and means of suppressing the bitter response to theseligands.

The universe of compounds that provoke a bitter response in humansstructurally very diverse. Therefore, if research into bitter receptorsis to be of any practical significance to the food and pharmaceuticalindustries, all bitter receptors will need to be identified, and onceidentified, there has to be a rigorous understanding of how specificreceptors are matched to particular structural classes of bittercompounds. Unfortunately, although much basic research has beenconducted in the area of bitter taste receptors, there are potentiallymany more bitter receptors to be discovered, and little is still knownas to whether the known members of the human T2R family of bitterreceptors actually respond to bitter tastants, and if so what, if any,specificity they show to ligand substructures.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to bitter-taste receptors and their rolein bitter taste transduction. The invention also relates to assays forscreening molecules that modulate, e.g. suppress or block bitter tastetransduction, or enhance bitter taste response.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., screening forcompounds that inhibit bitter taste, will be apparent from the followingdescription, from the drawings and from the claims.

Surprisingly, applicant has now found a new group of putative bittertaste receptors, and in respect of certain known bitter receptors,applicant has found that they respond with specificity towards classesof bitter compounds that are important in the food and pharmaceuticalindustries.

In a first aspect of the invention there is provided a new group ofputative bitter receptors. The genetics of bitter tasting has beenextensively studied in mice and rats. Therefore, applicant compared thenucleotide sequences encoding polypeptides previously proposed to bebitter receptors with publicly available human nucleic acid sequences inthe NCBI database using the BLAST® search methodology (Parameters:Expect=0.01, Filter=default). Surprisingly, the search identified 24 DNAsequences (from human chromosomes 5, 7, and 12) that, because of theirhomology to a mouse nucleic acid sequence that encodes a polypeptide(T2R5) previously designated as a bitter receptor, we designated asbitter receptor-encoding. Bitter taste receptors were originallyassigned identifiers starting with the three characters “T2R”(identifying the receptor family) followed by a number (e.g., 1, 2, 3,etc.) that identifies a particular receptor, e.g., T2R5. More recently adifferent system has been used in which the identifiers start with fivecharacters “TAS2R” (identifying the receptor family) followed, aspreviously, by a number (e.g., 1, 2, 3, etc.) that identifies aparticular receptor, e.g., TAS2R5. A lower case letter in front of theidentifier indicates the species of the receptor (e.g., “h” for human,“r” for rat, and “m” for mouse). Thus, for example, mTAS2R5 is a mousebitter receptor and hTAS2R2 is a human bitter receptor. For consistencythe new TAS2R identifier system is used throughout the rest of thisapplication.

Of the 24 coding sequences identified by the search, 12 are believed tobe novel; the polypeptides encoded by these novel sequences aredesignated hTAS2R38-41, and 43-50. The DNA sequences encoding thepolypeptides are assigned SEQ ID NOs: 2 (hTAS2R38), 4 (hTAS2R39), 6(hTAS2R40), 8 (hTAS2R41), 10 (hTAS2R43), 12 (hTAS2R44), 14(hTAS2R45), 16(hTAS2R46), 18 (hTAS2R47), 20 (hTAS2R48), 22 (hTAS2R49), and 24(hTAS2R50), respectively, and the amino acid sequences of thepolypeptides are assigned SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,21, and 23, respectively.

The 12 additional (possibly novel) sequences identified by the searchencode polypeptides, which are designated hTAS2R1, 4, 5, 7-10, 13, 14,16, 3, 42 and 60. The DNA sequences encoding the polypeptides areassigned SEQ ID NOs: 26 (hTAS2R1), 28 (hTAS2R4), 30 (hTAS2R5), 32(hTAS2R7), 34 (hTAS2R8), 36 (hTAS2R9), 38 (hTAS2R10), 40 (hTAS2R13), 42(hTAS2R14), 44 (hTAS2R16), 46 (hTAS2R3), 48 (hTAS2R42), and 50(hTAS2R60), respectively, and the amino acid sequences of thepolypeptides are assigned SEQ ID NOs: 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47 and 49, respectively.

Thus, one aspect of the present invention is a polynucleotide selectedfrom the group consisting of

-   (a) polynucleotides encoding at least the mature form of the    polypeptide having the deduced amino acid sequence as shown in SEQ    ID NOs 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23;-   (b) polynucleotides having the coding sequence, as shown in SEQ ID    NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 encoding at    least the mature form of the polypeptide;-   (c) polynucleotides encoding a fragment or derivative of a    polypeptide encoded by a polynucleotide of any one of (a) to (b),    wherein in said derivative one or more amino acid residues are    conservatively substituted compared to said polypeptide, and said    fragment or derivative has bitter substance binding activity;-   (d) polynucleotides which are at least 50% identical to a    polynucleotide as defined in any one of (a) to (c) and which code    for a polypeptide having bitter substance binding activity; and-   (e) polynucleotides the complementary strand of which hybridizes,    preferably under stringent conditions to a polynucleotide as defined    in any one of (a) to (d) and which code for a polypeptide having    bitter substance binding activity;    or the complementary strand of such a polynucleotide.

A polypeptide that exhibits bitter substance binding activity is apolypeptide that has at least 20% (e.g., at least: 20%; 30%; 40%; 50%;60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or even more) of theability of the respective full-length TAS2R to bind to a given bittersubstance. Binding assays and bitter substances are described hereinbelow.

In a preferred embodiment the polynucleotide of the present inventionencodes a polypeptide that still exhibits essentially the same activityas the respective mature bitter taste receptor, i.e. has “bitter tastereceptor activity”. Preferably the polypeptide has at least 20% (e.g.,at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%;or 100% or even more) of the ability of the respective full-length TAS2Rto release intracellular calcium in a heterologous cell expressionsystem like, for example, HEK293/15-cells, which stably express thealpha-subunit of promiscuous G-proteins, e.g. the mouse G₁₅ subunit, inresponse to bitter tastants, which is dependent on the expression ofpolypeptides encoded by the polynucleotides of the present invention.The amount of intracellular calcium release can be monitored by, forexample, the in vitro FLIPR assay described herein below.

The TAS2R nucleic acid molecules of the invention can be DNA, cDNA,genomic DNA, synthetic DNA, or, RNA, and can be double-stranded orsingle-stranded, the sense and/or an antisense strand. Segments of thesemolecules are also considered within the scope of the invention, and canbe produced by, for example, the polymerase chain reaction (PCR) orgenerated by treatment with one or more restriction endonucleases. Aribonucleic acid (RNA) molecule can be produced by in vitrotranscription.

The polynucleotide molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptides with SEQ ID NOs:1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, and 23). In addition, these nucleic acidmolecules are not limited to coding sequences, e.g., they can includesome or all of the non-coding sequences that lie upstream or downstreamfrom a coding sequence.

The polynucleotide molecules of the invention can be synthesized invitro (for example, by phosphoramidite-based synthesis) or obtained froma cell, such as the cell of a bacteria mammal. The nucleic acids can bethose of a human but also derived from a non-human primate, mouse, rat,guinea pig, cow, sheep, horse, pig, rabbit, dog, or cat as long as theyfulfill the criteria set out above. Combinations or modifications of thenucleotides within these types of nucleic acids are also encompassed.

In addition, the isolated nucleic acid molecules of the inventionencompass segments that are not found as such in the natural state.Thus, the invention encompasses recombinant nucleic acid moleculesincorporated into a vector (for example, a plasmid or viral vector) orinto the genome of a heterologous cell (or the genome of a homologouscell, at a position other than the natural chromosomal location).Recombinant nucleic acid molecules and uses therefore are discussedfurther below.

A polynucleotide belonging to a family of any of the TAS2R disclosedherein or a protein can be identified based on its similarity to therelevant TAS2R gene or protein, respectively. For example, theidentification can be based on sequence identity. In certain preferredembodiments the invention features isolated nucleic acid molecules whichare at least 50% (or 55%, 65%, 75%, 85°/a, 95%, or 98%) identical to:(a) a nucleic acid molecule that encodes the polypeptide of SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23; (b) the nucleotidesequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24;and (c) a nucleic acid molecule which includes a segment of at least 30(e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300,400, 500, 600, 700, 800, 850, 900, 950, 1000, or 1010) nucleotides ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24.

The determination of percent identity between two sequences isaccomplished using the mathematical algorithm of Karlin and Altschul,Proc. Natl. Acad. Sci. USA 90, 5873-5877, 1993. Such an algorithm isincorporated into the BLASTN and BLASTP programs of Altschul et al.(1990) J. Mol. Biol. 215, 403-410. BLAST nucleotide searches areperformed with the BLASTN program, score=100, wordlength=12, to obtainnucleotide sequences homologous to HIN-1-encoding nucleic acids. BLASTprotein searches are performed with the BLASTP program, score=50,wordlength=3, to obtain amino acid sequences homologous to the TAS2Rpolypeptide. To obtain gapped alignments for comparative purposes,Gapped BLAST is utilized as described in Altschul et al. (1997) NucleicAcids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs.

Hybridization can also be used as a measure of homology between twonucleic acid sequences. A nucleic acid sequence encoding any of theTAS2R disclosed herein, or a portion thereof, can be used as ahybridization probe according to standard hybridization techniques. Thehybridization of a TAS2R probe to DNA or RNA from a test source (e.g., amammalian cell) is an indication of the presence of the relevant TAS2RDNA or RNA in the test source. Hybridization conditions are known tothose skilled in the art and can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderatehybridization conditions are defined as equivalent to hybridization in2× sodium chloride/sodium citrate (SSC) at 30° C., followed by a wash in1×SSC, 0.1% SDS at 50° C. Highly stringent conditions are defined asequivalent to hybridization in 6× sodium chloride/sodium citrate (SSC)at 45° C., followed by a wash in 0.2×SSC, 0.1% SDS at 65° C.

An “isolated DNA” is either (1) a DNA that contains sequence notidentical to that of any naturally occurring sequence, or (2), in thecontext of a DNA with a naturally-occurring sequence (e.g., a cDNA orgenomic DNA), a DNA free of at least one of the genes that flank thegene containing the DNA of interest in the genome of the organism inwhich the gene containing the DNA of interest naturally occurs. The termtherefore includes a recombinant DNA incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote. The term also includes a separate molecule suchas a cDNA where the corresponding genomic DNA has introns and thereforea different sequence; a genomic fragment that lacks at least one of theflanking genes; a fragment of cDNA or genomic DNA produced by polymerasechain reaction (PCR) and that lacks at least one of the flanking genes;a restriction fragment that lacks at least one of the flanking genes; aDNA encoding a non-naturally occurring protein such as a fusion protein,mutein, or fragment of a given protein; and a nucleic acid which is adegenerate variant of a cDNA or a naturally occurring nucleic acid. Inaddition, it includes a recombinant nucleotide sequence that is part ofa hybrid gene, i.e., a gene encoding a non-naturally occurring fusionprotein. It will be apparent from the foregoing that isolated DNA doesnot mean a DNA present among hundreds to millions of other DNA moleculeswithin, for example, cDNA or genomic DNA libraries or genomic DNArestriction digests in, for example, a restriction digest reactionmixture or an electrophoretic gel slice.

A further aspect of the present invention is a vector containing thepolynucleotide(s) of the present invention or a protein encoded by apolynucleotide of the present invention. The term “vector” refers to aprotein or a polynucleotide or a mixture thereof which is capable ofbeing introduced or of introducing the proteins and/or nucleic acidcomprised into a cell. It is preferred that the proteins encoded by theintroduced polynucleotide are expressed within the cell uponintroduction of the vector.

In a preferred embodiment the vector of the present invention comprisesplasmids, phagemids, phages, cosmids, artificial mammalian chromosomes,knock-out or knock-in constructs, viruses, in particular adenoviruses,vaccinia viruses, attenuated vaccinia viruses, canary pox viruses,lentiviris (Chang, L. J. and Gay, E. F. (20001) Curr. Gene Therap.1:237-251), herpes viruses, in particular Herpes simplex virus (HSV-1,Carlezon, W. A. et al. (2000) Crit. Rev. Neurobiol.), baculovirus,retrovirus, adeno-associated-virus (AAV, Carter, P. J. and Samulski, R.J. (2000) J. Mol. Med. 6:17-27), rhinovirus, human immune deficiencyvirus (HIV), filovirus and engineered versions thereof (see, forexample, Cobillger G. P. et al (2001) Nat. Biotechnol. 19:225-30),virosomes, “naked” DNA liposomes, and nucleic acid coated particles, inparticular gold spheres. Particularly preferred are viral vectors likeadenoviral vectors or retroviral vectors (Lindemann et al. (1997) Mol.Med. 3:466-76 and Springer et al. (1998) Mol. Cell. 2:549-58). Liposomesare usually small unilamellar or multilamellar vesicles made ofcationic, neutral and/or anionic lipids, for example, by ultrasoundtreatment of liposomal suspensions. The DNA can, for example, beionically bound to the surface of the liposomes or internally enclosedin the liposome. Suitable lipid mixtures are known in the art andcomprise, for example, DOTMA(1,2-Dioleyloxpropyl-3-trimethylammoniumbromid) and DPOE(Dioleoylphosphatidyletlhanolamin) which both have been used on avariety of cell lines.

Nucleic acid coated particles are another means for the introduction ofnucleic acids into cells using so called “gene guns”, which allow themechanical introduction of particles into the cells. Preferably theparticles itself are inert, and therefore, are in a preferred embodimentmade out of gold spheres.

In a further aspect the polynucleotide of the present invention isoperatively linked to expression control sequences allowing expressionin prokaryotic and/or eukaryotic host cells. Thetranscriptional/translational regulatory elements referred to aboveinclude but are not limited to inducible and non-inducible,constitutive, cell cycle regulated, metabolically regulated promoters,enhancers, operators, silencers, repressors and other elements that areknown to those skilled in the art and that drive or otherwise regulategene expression. Such regulatory elements include but are not limited toregulatory elements directing constitutive expression like, for example,promoters transcribed by RNA polymerase III like, e.g., promoters forthe snRNA U6 or snRNA 7SK gene, the cytomegalovirus hCMV immediate earlygene, the early or late promoters of SV40 adenovirus, viral promoter andactivator sequences derived from, e.g., NBV, HCV, HSV, HPV, EBV, HTLV,MMTV or HIV; which allow inducible expression like, for example, CUP-1promoter, the tet-repressor as employed, for example, in the tet-on ortet-off systems, the lac system, the trp system; regulatory elementsdirecting tissue specific expression, preferably taste bud specificexpression, e.g., PLCβ2 promoter or gustducin promoter, regulatoryelements directing cell cycle specific expression like, for example,cdc2, cdc25C or cyclin A; or the TAC system, the TRC system, the majoroperator and promoter regions of phage A, the control regions of fd coatprotein, the promoter for 3-phosphoglycerate kinase, the promoters ofacid phosphatase, and the promoters of the yeast α- or a-mating factors.

As used herein, “operatively linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

Similarly, the polynucleotides of the present invention can form part ofa hybrid gene encoding additional polypeptide sequences, for example, asequence that functions as a marker or reporter. Examples of marker andreporter genes include β-lactamase, chloramphenicol acetyltransferase(CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo^(r), G418^(r)), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example,additional sequences that can serve the function of a marker orreporter. Generally, the hybrid polypeptide will include a first portionand a second portion; the first portion being a TAS2R polypeptide andthe second portion being, for example, the reporter described above oran Ig constant region or part of an Ig constant region, e.g., the CH₂and CH3 domains of IgG2a heavy chain. Other hybrids could include anantigenic tag or His tag to facilitate purification and/or detection.Recombinant nucleic acid molecules can also contain a polynucleotidesequence encoding a TAS2R polypeptide operatively linked to aheterologous signal sequence. Such signal sequences can direct theprotein to different compartments within the cell and are well known tosomeone of skill in the art. A preferred signal sequence is a sequencethat facilitates secretion of the resulting protein.

Another aspect of the present invention is a host cell geneticallyengineered with the polynucleotide or the vector as outlined above. Thehost cells that may be used for purposes of the invention include butare not limited to prokaryotic cells such as bacteria (for example, E.coli and B. subtilis), which can be transformed with, for example,recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expressionvectors containing the polynucleotide molecules of the invention; simpleeukaryotic cells like yeast (for example, Saccharomyces and Pichia),which can be transformed with, for example, recombinant yeast expressionvectors containing the polynucleotide molecule of the invention; insectcell systems like, for example, Sf9 of Hi5 cells, which can be infectedwith, for example, recombinant virus expression vectors (for example,baculovirus) containing the polynucleotide molecules of the invention;Xenopus oocytes, which can be injected with, for example, plasmids;plant cell systems, which can be infected with, for example, recombinantvirus expression vectors (for example, cauliflower mosaic virus (CaMV)or tobacco mosaic virus (TMV)) or transformed with recombinant plasmidexpression vectors (for example, Ti plasmid) containing a TAS2Rnucleotide sequence; or mammalian cell systems (for example, COS, CHO,BHK, HEK293, VERO, HeLa, MDCK, Wi38, and NIH 3T3 cells), which can betransformed with recombinant expression constructs containing, forexample, promoters derived, for example, from the genome of mammaliancells (for example, the metallothionein promoter) from mammalian viruses(for example, the adenovirus late promoter and the vaccinia virus 7.5Kpromoter) or from bacterial cells (for example, the tet-repressorbinding its employed in the tet-on and tet-off systems). Also useful ashost cells are primary or secondary cells obtained directly from amammal and transfected with a plasmid vector or infected with a viralvector. Depending on the host cell and the respective vector used tointroduce the polynucleotide of the invention the polynucleotide canintegrate, for example, into the chromosome or the mitochondrial DNA orcan be maintained extrachromosomally like, for example, episomally orcan be only transiently comprised in the cells.

In a preferred embodiment, the TAS2R encoded by the polynucleotides ofthe present invention and which are expressed by such cells arefunctional, i.e., upon binding to one or more bitter molecules theytrigger an activation pathway in the cell. The cells are preferablymammalian (e.g., human, non-human primate, horse, bovine, sheep, goat,pig, dog, cat, goat, rabbit, mouse, rat, guinea pig, hamster, or gerbil)cells, insect cells, bacterial cells, or fungal (including yeast) cells.

A further aspect of the present invention is a transgenic non-humananimal containing a polynucleotide, a vector and/or a host cell asdescribed above. The animal can be a mosaic animal, which means thatonly part of the cells making up the body comprise polynucleotides,vectors, and/or cells of the present invention or the animal can be atransgenic animal which means that all cells of the animal comprise thepolynucleotides and/or vectors of the present invention or are derivedfrom a cell of the present invention. Mosaic or transgenic animals canbe either homo- or heterozygous with respect to the polynucleotides ofthe present invention contained in the cell. In a preferred embodimentthe transgenic animals are either homo- or heterozygous knock-out orknock-in animals with respect to the genes which code for the proteinsof the present invention. The animals can in principal be any animal,preferably, however, it is a mammal, selected from the group ofnon-human primate horse, bovine, sheep, goat, pig, dog, cat, goat,rabbit, mouse, rat, guinea pig, hamster, or gerbil.

Another aspect of the present invention is a process for producing apolypeptide encoded by a polynucleotide of the present inventioncomprising: culturing the host cell described above and recovering thepolypeptide encoded by said polynucleotide. Preferred combinations ofhost cells and vectors are outlined above and further combination willbe readily apparent to someone of skill in the art. Depending on theintended later use of the recovered peptide a suitable cell type can bechosen. Eukaryotic cells are preferably chosen, if it is desired thatthe proteins produced by the cells exhibit an essentially naturalpattern of glycosylation and prokaryotic cells are chosen, if, forexample, glycosylation or other modifications, which are normallyintroduced into proteins only in eukaryotic cells, are not desired ornot needed.

A further aspect of the invention is a process for producing cellscapable of expressing at least one of the bitter taste receptorpolypeptides comprising genetically engineering cells in vitro with atleast one of the vectors described above, wherein said bitter tastereceptor polypeptide(s) is(are) encoded by a polynucleotide of thepresent invention.

Another aspect of the invention is a polypeptide having the amino acidsequence encoded by a polynucleotide of the invention or obtainable bythe process mentioned above. The polypeptides of the invention includeall those disclosed herein and functional fragments of thesepolypeptides. “Polypeptide” and “protein” are used interchangeably andmean any peptide-linked chain of amino acids, regardless of length orposttranslational modification. As used herein, a functional fragment ofa TAS2R is a fragment of the TAS2R that is shorter than the full-lengthTAS2R but that has at least 20% (e.g., at least: 20%; 30%; 40%; 50%;60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or even more) of theability of the full-length TAS2R to bind to a bitter substance to whichthe full-length TAS2R binds. Binding assays and bitter substances aredescribed herein. Further bitter substances can be identified by thebinding assays and bitter taste receptor activity assays describedherein. The polypeptides embraced by the invention also include fusionproteins that contain either a full-length TAS2R polypeptide or afunctional fragment of it fused to an unrelated amino acid sequence. Theunrelated sequences can be additional functional domains or signalpeptides. Signal peptides are described in greater detail andexemplified below.

The polypeptides can be any of those described above but with not morethan 50 (e.g., not more than: 50, 45, 40, 35, 30, 25, 20, 15, 14, 13,12, 11, 10, nine, eight, seven, six, live, four, three, two, or one)conservative substitutions. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine,glutamine, serine and threonine; lysine, histidine and arginine; andphenylalanine and tyrosine. All that is required of a polypeptide havingone or more conservative substitutions is that it has at least 20%(e.g., at least: 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%;99.5%; or 100% or even more) of the ability of the wild-type,full-length TAS2R to bind to a bitter substance, preferably the abilityto release intracellular calcium, when expressed in a cellular system.

The polypeptides can be purified from natural sources (e.g., blood,serum, plasma, tissues or cells such as normal tongue cells or any cellthat naturally produces the relevant TAS2R polypeptides). Smallerpeptides (less than 50 amino acids long) can also be convenientlysynthesized by standard chemical means. In addition, both polypeptidesand peptides can be produced by standard in vitro recombinant DNAtechniques and in vivo transgenesis, using nucleotide sequences encodingthe appropriate polypeptides or peptides. Methods well-known to thoseskilled in the art can be used to construct expression vectorscontaining relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example, thetechniques described in Sambrook et al., Molecular Cloning: A LaboratoryManual (2nd Ed.) [Cold Spring Harbor laboratory, N.Y., 1989], andAusubel et al., Current Protocols in Molecular Biology [Green PublishingAssociates and Wiley Interscience, N.Y., 1989].

Polypeptides and fragments of the invention also include those describedabove, but modified for in vivo use by the addition, at the amino-and/or carboxyl-terminal ends, of blocking agents to facilitate survivalof the relevant polypeptide in vivo. This can be useful in thosesituations in which the peptide termini tend to be degraded by proteasesprior to cellular uptake. Such blocking agents can include, withoutlimitation, additional related or unrelated peptide sequences that canbe attached to the amino and/or carboxyl terminal residues of thepeptide to be administered. This can be done either chemically duringthe synthesis of the peptide or by recombinant DNA technology by methodsfamiliar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptides can be covalently or noncovalently coupled topharmaceutically acceptable “carrier” proteins prior to administration.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of the functional peptides or peptidefragments. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to bind to a bitter compound in a manner qualitativelyidentical to that of the TAS2R functional fragment from which thepeptidomimetic was derived. Peptidomimetic compounds can have additionalcharacteristics that enhance their therapeutic utility, such asincreased cell permeability and prolonged biological half-life.

The peptidomimetics typically have a backbone that is partially orcompletely nonpeptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

The term “isolated” polypeptide or peptide fragment as used hereinrefers to a polypeptide or a peptide fragment which either has nonaturally-occulting counterpart or has been separated or purified fromcomponents which naturally accompany it, e.g., in tissues such astongue, pancreas, liver, spleen, ovary, testis, muscle, joint tissue,neural tissue, gastrointestinal tissue or tumor tissue, or body fluidssuch as blood, serum, or urine. Typically, the polypeptide or peptidefragment is considered “isolated” when it is at least 70%, by dryweight, free from the proteins and other naturally-occurring organicmolecules with which it is naturally associated. Preferably, apreparation of a polypeptide (or peptide fragment thereof) of theinvention is at least 80%, more preferably at least 90%, and mostpreferably at least 99%, by dry weight, the polypeptide (or the peptidefragment thereof), respectively, of the invention. Thus, for example, apreparation of polypeptide x is at least 80%, more preferably at least90%, and most preferably at least 99%, by dry weight, polypeptide x.Since a polypeptide that is chemically synthesized is, by its nature,separated from the components that naturally accompany it, the syntheticpolypeptide is “isolated.”

An isolated polypeptide (or peptide fragment) of the invention can beobtained, for example, by extraction from a natural source (e.g., fromtissues or bodily fluids); by expression of a recombinant nucleic acidencoding the polypeptide; or by chemical synthesis. A polypeptide thatis produced in a cellular system different from the source from which itnaturally originates is “isolated,” because it will necessarily be freeof components which naturally accompany it. The degree of isolation orpurity can be measured by any appropriate method, e.g., columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

A further aspect of the invention is an antibody, which specificallybinds to the polypeptide encoded by a polynucleotide of the invention orobtainable by the process mentioned above. The term “antibody” comprisesmonoclonal and polyclonal antibodies and binding fragments thereof, inparticular Fc-fragments as well as so called “single-chain-antibodies”(Bird R. E. et al (1988) Science 242:423-6), chimeric, humanized, inparticular CDR-grafted antibodies, and dia or tetrabodies (Holliger P.et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-8). Also comprisedare immunoglobulin like proteins that are selected through techniquesincluding, for example, phage display to specifically bind to thepolypeptides of the present invention. Preferred antibodies bind to theextracellular domain of bitter receptors and in particular to thosedomains responsible for binding to bitter tastants.

In yet another embodiment there is provided a molecule, or collectionsof molecules containing a molecule, that act to antagoniseaforementioned receptors in particular the bitter taste response, andmethods for screening for such molecules.

Therefore, a further aspect of the invention is a nucleic acid moleculewhich specifically hybridizes to a polynucleotide of the presentinvention. In particular this nucleic acid molecule is an inhibitingRNA. Preferred inhibiting RNAs are antisense constructs hybridizing to apolynucleotide of the present invention, RNAi, siRNA or a ribozyme. Thedesign of such inhibiting RNAs would be readily apparent to someone ofskill in the art.

Another type of antagonist/inhibitor against the polypeptides of thepresent invention is an antibody, which is preferably directed againstthe extracellular domain of the respective bitter taste receptor andeven more preferably binds to the site(s) of the receptor thatinteract(s) with the bitter substance(s) essentially without triggeringthe release of intracellular calcium. Further antagonists to the bittertaste response of a receptor are fragments of the receptor which havethe capability to bind to the bitter substances as defined above. Suchfragments can bind to the bitter substance and, thus, competitivelyantagonize the activity of the respective TAS2R. If such antagonistsare, for example, employed within foodstuff to suppress the bitter tasteof a specific bitter substance they might be exposed to a proteolyticenvironment and in this case the modifications of the polypeptidesoutlined above could be used to stabilize the competitive bitterreceptor antagonist. However, various additional modifications, whichstabilize such fragments will be readily apparent to the skilled person.

Antagonists and agonists of the bitter taste receptors described hereinare of great importance for specific stimulation of a given bitter tastereceptor or to antagonize it. The bitter taste response of the receptoris elicited by the specific binding of the respective bitter substance.Therefore, the present invention is also directed at a process forisolating a compound that binds to a polypeptide encoded by apolynucleotide selected from the group consisting of:

-   (a) polynucleotides encoding at least the mature form of the    polypeptide having the deduced amino acid sequence as shown in SEQ    ID NOs1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,    35, 37, 39, 41, 43, 45, 47 and 49;-   (b) polynucleotides having the coding sequence, as shown in SEQ ID    NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 48 and 50 encoding at least the mature form    of the polypeptide;-   (c) polynucleotides encoding a fragment or derivative of a    polypeptide encoded by a polynucleotide of any one of (a) to (b),    wherein in said derivative one or more amino acid residues are    conservatively substituted compared to said polypeptide, and said    fragment or derivative has bitter substance binding activity;-   (d) polynucleotides which are at least 50% identical to a    polynucleotide as defined in any one of (a) to (c) and which code    for a polypeptide having bitter substance binding activity; and-   (e) polynucleotides the complementary strand of which hybridizes,    preferably under stringent conditions to a polynucleotide as defined    in any one of (a) to (d) and which code for a polypeptide having    bitter substance binding activity;    comprising:-   (1) contacting said polypeptide or a host cell genetically    engineered with said polynucleotide or with a vector containing said    polynucleotide with a compound;-   (2) detecting the presence of the compound which binds to said    polypeptide; and-   (3) determining whether the compound binds said polypeptide.

A polynucleotide employed in this process is in preferred embodiments ofthe invention at least 50% (or 55%, 65%, 75%, 85%/a, 95%, or 98%)identical to: (a) a nucleic acid molecule that encodes the polypeptideof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47 or 49; (b) the nucleotide sequence ofSEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48 or 50; and has a length of at least 30(e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300,400, 500, 600, 700, 800, 850, 900, 950, 1000, or 1010) of thenucleotides of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 50.

Furthermore for all of the above described hTAS2Rs, which can beemployed in a process for isolating binding compounds, with theexception of hTAS2R40, single nucleotide polymorphisms are known. 79 ofthese are listed in Table I below. 61 of those result in an amino acidchange. Polynucleotides or polypeptides that differ from therespectively in SEQ ID 1-50 indicated sequences by the nucleotide andamino acid change as indicted in Table I can similarly be employed forthe process of the present invention.

TABLE I Gen + Substitution Position Acession Name of Amino Base AminoAllelic No. SNP Base acid pair acid frequency hTAS2R1 rs2234231 C/T P/L128 43 unknown NM019599 rs41469 G/A R/H 332 111 A 0.46/G 0.54 rs223432G/A C/Y 422 141 unknown rs2234233 C/T R/W 616 206 C 0.87/T 0.13rs2234234 C/T S/S 675 225 unknown rs2234235 T/C L/L 850 284 unknownhTAS2R3 rs227009 C/T G/G 807 369 unknown NM016943 hTAS2R4 ss3181498 G/AR/Q 8 3 unknown NM016944 rs2233996 G/C R/R 9 3 unknown rs2233997 A/C Y/C17 6 unknown rs2233998 T/C F/S 20 7 unknown rs2233999 T/A F/L 186 62unknown rs2234000 C/T T/M 221 74 C 0.94/T 0.56 rs2234001 G/C V/L 286 96C 0.78/G 0.22 rs2234002 G/A S/N 512 171 A 0.78/G 0.22 rs2234003 A/G I/V571 191 unknown hTAS2R5 rs2234013 G/A G/S 58 20 unknown NM018980rs2227264 G/T S/I 77 26 unknown rs2234014 C/T P/L 338 113 unknownrs2234015 G/A R/Q 638 213 unknown rs2234016 G/T R/L 294 881 unknownhTAS2R7 rs3759251 A/T T/S 787 263 A 0.97/T 0.03 NM023919 rs3759252 C/AI/I 828 276 unknown rs619381 G/A M/I 912 304 unknown hTAS2R8 ss2391467G/A L/L 549 183 unknown NM023918 rs2537817 A/G M/V 922 308 unknownhTAS2R9 rs3741845 T/C V/A 560 187 C 073/T 027 NM23917 rs3944035 C/T L/F910 304 unknown rs2159903 C/T P/L 926 309 unknown hTAS2R10 rs597468 C/TT/M 467 156 unknown NM23921 hTAS2R13 ss1478988 A/G N/S 776 259 C 0.73/T0.27 NM23920 hTAS2R14 rs3741843 G/A R/R 375 125 A 0.97/G 0.03 NM23922hTAS2R16 rs2233988 C/T T/T 300 100 unknown NM016945 rs2692396 G/C V/V303 101 unknown rs2233989 T/C L/L 460 154 unknown rs846664 T/G N/K 516172 A 0.71/C 0.29 rs860170 G/A R/H 665 222 A 0.55/G 0.45 hTAS2R38 PTCPaper G/A V/I 886 296 G 0.38/A 0.62 AF494321 rs1726866 T/C V/A 785 262 G0.38/T 0.62 rs713598 C/T A/P 49 145 C 0.36/G 0.64 hTAS2R38 SNP1 A/T N/I557 186 C 0.60/G 0.40 hTAS2R39 hTAS2R39 SNP1 A/AA frameshift 967 323unknown AF494230 hTAS2R41 rs1404635 A/G T/T 189 64 unknown AF494232hTAS2R41 SNP1 T/C L/P 380 127 unknown hTAS2R41 SNP2 A/G S/S 885 295unknown hTAS2R42 rs1650017 G/C A/P 931 311 unknown AX097739 rs1669411T/C N/N 930 310 unknown rs1669412 G/A R/Q 875 292 unknown rs1451772 A/GY/C 794 265 unknown rs1669413 G/T G/W 763 255 unknown rs1650019 A/G L/L561 187 unknown hTAS2R43 rs3759246 G/C R/T 893 298 unknown AF494237hTAS2R43 SNP1 C/G S/W 104 35 unknown hTAS2R43 SNP2 G/A R/H 635 212unknown hTAS2R43 SNP3 G/C T/T 663 221 unknown hTAS2R44 rs3759247 G/AW/stop 900 300 unknown AF494228 rs3759246 G/C R/T 893 298 unknownhTAS2R44 SNP1 A/T M/L 162 484 unknown hTAS2R44 SNP2 T/A F/Y 869 290unknown hTAS2R44 SNP3 G/A V/M 899 297 unknown hTAS2R45 rs3759247 A/GG/stop 900 300 unknown AF494226 rs3759246 G/C R/T 893 298 unknownrs3759245 C/T R/C 712 238 unknown rs3759244 T/C F/L 703 235 unknownhTAS2R46 rs2708381 G/A W/stop 749 250 unknown AF494227 rs2708380 T/A L/M682 228 unknown rs2598002 T/G F/V 106 36 unknown hTAS2R46 SNP1 A/T Q/H888 296 unknown hTAS2R46 SNP2 A/G M/V 889 297 unknown hTAS2R46 SNP3 T/CF/F 108 36 unknown hTAS2R47 rs2597924 G/A R/H 920 307 unknown AF494233rs1669405 T/G L/W 842 281 unknown rs2599404 T/G F/L 756 252 unknownrs2600355 T/G V/V 54 18 unknown hTAS2R48 rs1868769 T/C L/L 418 140unknown AF494234 hTAS2R49 hTAS2R49 A/G K/R 164 55 unknown AF494236 SNP1hTAS2R50 rs1376521 A/G Y/C 608 203 G 0.66/A 0.34 AF494235 hTAS2R50 SNP1A/G P/P 777 259 unknown

A polypeptide that exhibits bitter substance binding activity is apolypeptide that has at least 20% (e.g., at least: 20%; 30%; 40%; 50%;60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or even more) of theability of the respective full-length TAS2R to bind to a given bittersubstance. Binding assays and bitter substances are described herein.

The term “contacting” in the context of the present invention means anyinteraction between the compound with the polypeptide of the invention,whereby any of the at least two components can be independently of eachother in a liquid phase, for example in solution, or in suspension orcan be bound to a solid phase, for example, in the form of anessentially planar surface or in the form of particles, pearls or thelike. In a preferred embodiment a multitude of different compounds areimmobilized on a solid surface like, for example, on a compound librarychip and the protein of the present invention is subsequently contactedwith such a chip. In another preferred embodiment the cells geneticallyengineered with the polynucleotide of the invention or with a vectorcontaining such a polynucleotide express the bitter taste receptor atthe cell surface and are contacted separately in small containers, e.g.,microtitre plates, with various compounds.

Detecting the presence and the binding of the compound to thepolypeptide can be carried out, for example, by measuring a marker thatcan be attached either to the protein or to the compound. Suitablemarkers are known in the art and comprise, for example, fluorescence,enzymatic or radioactive markers. The binding of the two components can,however, also be measured by the change of an electrochemical parameterof the binding compound or of the protein, e.g. a change of the redoxproperties of either the protein or the binding compound, upon binding.Suitable methods of detecting such changes comprise, for example,potentiometric methods. Further methods for detecting and/or measuringthe binding of the two components to each other are known in the art andcan without limitation also be used to measure the binding of thecompound to the polypeptide. The effect of the binding of the compoundon the activity of the polypeptide can also be measured by assessingchanges in the cells that express the polypeptides, for example, byassaying the intracellular release of calcium upon binding of thecompound.

As a further step after measuring the binding of a compound and afterhaving measured the binding strength of at least two different compoundsat least one compound can be selected, for example, on grounds of ahigher binding strength or on grounds of the detected intracellularrelease of calcium.

The thus selected compound is than in a preferred embodiment modified ina further step. Modification can be effected by a variety of methodsknown in the art, which include without limitation the introduction ofone or more, preferably two, three or four novel side chains or residuesor the exchange of one or more functional groups like, for example,introduction or exchange of halogens, in particular F, Cl or Br; theintroduction or exchange of lower alkyl residues, preferably having oneto five carbon atoms like, for example, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl or iso-pentylresidues; lower alkenyl residues, preferably having two, three, four orfive carbon atoms; lower alkinyl residues, preferably having two, three,four or five carbon atoms, which can in a preferred embodiment befurther substituted with F, Cl, Br, —NH₂, NO₂, OH, SH, NH, CN, aryl,heteroaryl, COH or COOH group; or the introduction of, for example, oneor more residue(s) selected from the group consisting of NH₂, NO₂, OH,SH, NH, CN, aryl, alkylaryl, heteroaryl, alkylheteroaryl, COH or COOHgroup.

The thus modified binding substances are than individually tested withthe method of the present invention, i.e. they are contacted with thepolypeptide as such or with the polypeptide expressed in a cell, andsubsequently binding of the modified compounds is measured. In this stepboth the binding per se can be measured and/or the effect of thefunction of the protein like, e.g. the intracellular calcium release. Ifneeded the steps of selecting the compound, modifying the compound,contacting the compound with a polypeptide of the invention andmeasuring the binding of the modified compound to the polypeptide can berepeated a third or any given number of times as required. The abovedescribed method is also termed “directed evolution” of the compoundsince it involves a multitude of steps including modification andselection, whereby binding compounds are selected in an “evolutionary”process optimizing their capabilities with respect to a particularproperty, e.g. its binding activity, its ability to activate, inhibit ormodulate the activity, in particular inhibit the intracellular releaseof calcium mediated by the polypeptides of the present invention.

Of particular interest are compounds that antagonize the bitter tastereceptor activity of the TAS2Rs disclosed and described herein. Thespecification thereby enables the skilled person to design intelligentcompound libraries to screen for antagonists to the bitter response ofthese receptors, which in turn enables the development of compounds andcompositions to suppress or eliminate bitter tasting components offoods, in particular animal foods, nutrients and dietary supplements andpharmaceutical or homeopathic preparations containing suchphyto-chemicals. Similarly, the invention also enables the skilledperson to screen for additional bitter ligands, or even to screen forcompounds that enhance a bitter response, such as might be useful in thefood industry. Therefore, another aspect of the invention is a processfor isolating an antagonist of the bitter taste receptor activity of thepolypeptide encoded by a polynucleotide selected from the groupconsisting of:

-   (a) polynucleotides encoding at least the mature form of the    polypeptide having the deduced amino acid sequence as shown in SEQ    ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,    33, 35, 37, 39, 41, 43, 45, 47 and 49;-   (b) polynucleotides having the coding sequence, as shown in SEQ ID    NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,    36, 38, 40, 42, 44, 46, 48 and 50 encoding at least the mature form    of the polypeptide;-   (c) polynucleotides encoding a fragment or derivative of a    polypeptide encoded by a polynucleotide of any one of (a) to (b),    wherein in said derivative one or more amino acid residues are    conservatively substituted compared to said polypeptide, and said    fragment or derivative has bitter taste receptor activity;-   (d) polynucleotides which are at least 50% identical to a    polynucleotide as defined in any one of (a) to (c) and which code    for a polypeptide having bitter taste receptor activity; and-   (e) polynucleotides the complementary strand of which hybridizes,    preferably under stingent conditions to a polynucleotide as defined    in any one of (a) to (d) and which code for a polypeptide having    bitter taste receptor activity;    comprising:-   (1) contacting said polypeptide or a host cell genetically    engineered with said polynucleotide or with a vector containing said    polynucleotide with a potential antagonist;-   (2) determining whether the potential antagonists antagonizes the    bitter taste receptor activity of said polypeptide.

The polynucleotide employed in this process encodes a polypeptide thatstill exhibits essentially the same activity as the respective maturebitter taste receptor, i.e. has “bitter taste receptor activity”.Preferably the polypeptide has at least 20% (e.g., at least: 20%; 30%;40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or evenmore) of the activity of the respective full-length TAS2R. One preferredway of measuring TAS2R activity is the ability to release intracellularcalcium in a heterologous cell expression system like, for example,(HEK293/15) that stably expresses the alpha-subunit of promiscuousG-proteins, e.g. the mouse G₁₅ subunit or chimeric, in response tobitter tastants, which is dependent on the expression of polypeptidesencoded by the polynucleotides of the present invention. The amount ofintracellular calcium released can be monitored by, for example, the invitro FLIPR assay described herein but also by the measurement of one ofa variety of other parameters including, for example, IP₃ or cAMP.Additional ways of measuring G-protein coupled receptor activity areknown in the art and comprise without limitation electrophysiologicalmethods, transcription assays, which measure, e.g. activation orrepression of reporter genes which are coupled to regulatory sequencesregulated via the respective G-protein coupled signaling pathway, suchreporter proteins comprise, e.g., CAT or LUC; assays measuringinternalization of the receptor; or assays in frog melanophore systems,in which pigment movement in melanophores is used as a read out for theactivity of adenylate cyclase or phospholipase C (PLC), which in turnare coupled via G-proteins to exogenously expressed receptors (see, forexample, McClintock T. S. et al. (1993) Anal. Biochem. 209: 298-305;McClintock T. S., and Lerner M. R. (1997) Brain Res. Brain, Res. Protoc.2: 59-68, Potenza M N (1992) Pigment Cell Res. 5: 372-328, and PotenzaM. N. (1992) Anal. Biochem. 206: 315-322)

As described above with the exception of hTAS2R40, single nucleotidepolymorphisms are known for all of the above hTAS2Rs, which can beemployed in a process for isolating an antagonist of the bitter tastereceptor activity. Polynucleotides or polypeptides that differ from therespectively in SEQ ID 1-50 indicated sequences by the nucleotide andamino acid change as indicted in Table I can similarly be employed forthe process of the present invention.

The term “contacting” has the meaning, as outlined above. A potentialantagonist is a substance which lowers the respective bitter tastereceptor activity determined in the absence of the antagonist by atleast 10% (e.g., at least: 1%, 15% 20%; 30%; 40%; 50%; 60%; 70%; 80%;90%; 95%; 98%; 99%; 99.5%; or 100%) once contacted with the bitter tastereceptor.

In a preferred embodiment the process further comprises the contactingof the polypeptide with an agonist of the respective bitter tastereceptor activity. The contacting of the bitter taste receptor with theagonist can be carried out prior, concomitantly or after contacting thepolypeptide with the potential antagonist.

It has been demonstrated by the inventors that the bitter receptorshTAS2R10, hTAS2R14, hTAS2R16, hTAS2R38, hTAS2R43, hTAS2R44, hTAS2R45,hTAS2R46 and hTAS2R48 respond with specificity to (a) defined classe(s)of ligand(s) that include a class of useful phyto-chemicals in afunctional expression assay. Therefore, in an even more preferredembodiment the polypeptides and agonist employed together in aboveprocess are selected from the group consisting of:

-   (a) the polypeptide encoded by the polynucleotide outlined above as    determined by SEQ ID NO: 1 and SEQ ID NO: 2 and the agonist selected    from the group consisting of acetylthiourea,    N,N-dimethylthioformamide, N,N′-diphenylthiourea, N-ethylthiourea,    2-imidazolidinethione, 4(6)-methyl-2-thiouracil, N-methylthiourea,    phenylthio-carbamid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil,    tetramethylthiourea, thioacetamide, thioacetanilide,    2-thiobarbituric acid, and 2-thiouracil and functional derivatives    thereof;-   (b) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 9 and SEQ ID NO: 10 and the agonist    selected from the group consisting of saccharin and functional    derivatives thereof;-   (c) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 11 and SEQ ID NO: 12 and the agonist    selected from the group consisting of saccharin and acesulfame K and    functional derivatives thereof;-   (d) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 13 and SEQ ID NO: 14 and the agonist    selected from the group consisting of absinthine and functional    derivatives thereof;-   (e) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 15 and SEQ ID NO: 16 and the agonist    selected from the group consisting of absinthine and functional    derivatives thereof;-   (f) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 19 and SEQ ID NO: 20 and the agonist    selected from the group consisting of absinthine and functional    derivatives thereof;-   (g) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 37 and SEQ ID NO: 38 and the agonist    selected from the group consisting of strychnine, brucine,    denatonium benzoate, and absinthine and functional derivatives    thereof;-   (h) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 41 and SEQ ID NO: 42 and the agonist    selected from the group consisting of tyrosine, preferably    L-tyrosine, and other bitter tasting amino acids including, e.g.,    leucine, histidine phenylalanine and tryptophan, and functional    derivatives thereof; and-   (i) the polypeptide encoded by the polynucleotide of claim 1 or 2 as    determined by SEQ ID NO: 43 and SEQ ID NO: 44 and the agonist    selected from the group consisting of naphtyl-β-D-glucoside,    phenyl-β-D-glucoside, salicin, helicin, arbutin,    2-nitrophenylB-D-glucoside, 4-nitrophenyl-β-D-glucoside,    methyl-β-D-glucoside, esculin, 4-nitrophenyl-β-D-thioglucoside,    4-nitrophenyl-β-D-mannoside, and amygdalin and functional    derivatives thereof.

The term “functional derivatives thereof” refers to substances, whichare derived from the respectively indicated bitter substance by chemicalmodification and which elicit at least 20% (e.g., at least: 20%; 30%;40%; 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%; or 100% or evenmore) of the bitter taste receptor activity, if compared to therespective unmodified bitter substance. Chemical modification includeswithout limitation the introduction of one or more, preferably two,three or four novel side chains or residues or the exchange of one ormore functional groups like, for example, introduction or exchange of H;linear or branched alkyl, in particular lower alkyl (C₁, C₂, C₃, C₄, andC₅, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,tert-butyl, n-pentyl or iso-pentyl); substituted linear or branchedalkyl, in particular lower substituted alkyl; linear or branchedalkenyl, in particular lower alkenyl (C₂, C₃, C₄ and C₅, e.g. ethenyl,1-propenyl, 2-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl;substituted linear or branched alkenyl, in particular lower substitutedalkenyl; linear or branched alkinyl, in particular lower alkinyl (C₂,C₃, C₄ and C₅); substituted linear or branched alkinyl, in particularlower substituted alkinyl; linear or branched alkanol, in particularlower alkanol (C₁, C₂, C₃, C₄, and C₅); linear or branched alkanal, inparticular lower alkanal (C₁, C₂, C₃, C₄, and C₅, e.g. COH, CH₂COH,CH₂CH₂COH; aryl, in particular phenyl; substituted aryl, in particularsubstituted aryl; heteroaryl; substituted heteroaryl; alkylaryl, inparticular benzyl; substituted alkylaryl; in particular substitutedbenzyl; alkylheteroaryl; substituted alkylheteroaryl; aminoalkyl, C₁,C₂, C₃, C₄ and C₅, e.g. —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂; substitutedaminoalkyl; aminoketone, in particular —NHCOCH₃; substitutedaminoketone; aminoaryl, in particular —NH—Ph; substituted aminoaryl, inparticular substituted —NH—Ph; CN; NH₂; Halogen, in particular F, Cl,and Br; NO₂; OH; SH; NH; CN; or COOH group. If the residues mentionedabove are substituted they are preferably mono, di, or tri substitutedwith a substituent selected from the group of halogen, in particular F,Cl, and Br, NH₂, NO₂, OH, SH, NH, CN, aryl, alkylaryl, heteroaryl,alkylheteroaryl, COH or COOH.

In particular the hTAS2R16 receptor has been shown to respondspecifically to a narrow class of interesting phyto-chemicals selectedfrom the group consisting of bitter beta-glucopyranosides andmannopyranosides.

The beta-glucopyranosides and beta-mannosepyranosides are a group ofbitter compounds consisting of a hydrophobic residue attached to glucoseand mannose, respectively, by a beta-glycosidic bond.

Preferred compounds that bind to the bTAS2R16 taste receptor are chosenfrom beta glucopyranosides and beta-mannopyranosides defined by theformula:

These compounds were studied in vitro (see Table I and IV below) andalso by human panelists (see Table I below) as is described in greaterdetail below.

From these studies certain inferences can be drawn regarding theaffinity of the compounds towards activation of the hTAS2R16 receptor.Thus, for the promotion of activation the steric position at C2 can beeither alpha or beta and the beta-configuration of the glycosidic bondand the alpha steric position of the hydroxyl group at C4 of thepyranose ring are preferred. Whereas R can be hydrogen, it is preferredthat R is a substituent selected from C₁-C₈ alkyl which may be branched,linear or cyclic as appropriate; lower alkenyl residues, preferablyhaving two, three, four or five carbon atoms; lower alkinyl residues,preferably having two, three, four or five carbon atoms, which can in apreferred embodiment be further substituted with F, Cl, Br, NH₂, NO₂,OH, SH, NH, CN, aryl, heteroaryl, COH or COOH group; heteroaryl, e.g.benzofuran and cumarin; aryl, e.g. phenyl, naphtyl; or the same of othersugar residue, e.g. glucopyranoside, which itself can carry asubstituent R with the meaning as outlined above. Bulkier groups at Clmay increase the activation of the receptor. The aryl or heteroaryl maybe further substituted with one or more substituents. Preferredsubstituents of the aryl or heteroaryl group are F, Br, Cl, NO₂, loweralkyl with one, two, three, four, five, six, seven or eight carbon atomsand CH₂OH. The phenyl group is preferably mono, di, or trisubstituted inortho, para and/or meta position(s). The substituent at C6 is shown asan hydroxyl group above. However, the compounds activity as agonists arelittle effected by further or alternative substitution at this position,and there is design freedom at this part of the compound. Furthermore,without intending to be bound by theory, it is thought that thesubstituent “R” is not responsible for bitterness in these compounds.Rather, bitterness is thought to derive from a hydrogen acceptor anddonor site provided by two hydroxyl groups on the ring. In anotherembodiment the O-glycosidic bond of the compounds outlined above can bea S-glycosidic bond, as exemplified by the bitter substance4-nitrophenyl-β-D-thioglucoside.

Most preferred compounds are selected from the group consisting ofnaphtyl-β-D-glucoside, phenyl-β-D-glucoside, salicin, helicin, arbutin,2-nitrophenyl-β-D-glucoside, 4-nitrophenyl-β-D-glucoside,methyl-β-D-glucoside, esculin, 4-nitrophenyl-β-D-thioglucoside,4-nitrophenyl-β-D-mannoside, and amygdalin.

The beta-glucopyranosides are phytonutrients that represent an importantclass of compounds found in plant-derived foods that may be useful asdietary supplements, or in functional foods or medicaments for theprevention of disease states. However, due to their bitter after-tastethey are aversive to consumers and so they are routinely removed fromfoods during production and processing as is further described inDrewnowski, A. & Gomez-Carneros, C. Bitter taste, phytonutrients, andthe consumer: a review. Am. J. Clin. Nutr. 72, 1424-1435 (2000). Removalis laborious and therefore expensive. The alternative is to mask theoff-flavor using encapsulation technologies or organoleptic compounds asmasking agents. However, encapsulation technology may not be appropriatein pharmaceutics as this may affect the absorption characteristics ofthe active compound, whereas the use of masking agents may impart theirown characteristic flavor which may unbalance the flavor of food orbeverages.

Without wishing to be bound by any particular theory as to theirmechanism of action, applicant believes that the bitter receptorsactivate a G-protein and thereby initiate the aforementioned cellularactivation cascade as a result of conformational changes in the receptorafter binding by a ligand. Potential antagonists of the bitter responsewill contain functionality (i.e., will compete for binding at thereceptor, and/or act at another binding site through an allostericmechanism, and/or stabilize the receptor in the inactive conformation,and/or bind reversibly or irreversibly, and/or weaken receptor G proteininteraction, and/or interfere with G protein activation).

Similarly, in another embodiment of the invention, it has been foundthat the so-called hTAS2R10 receptor is activated by strychnine, andstrychnine analogues such as brucine as well as by denatonium benzoate,absinthine and other alkaloids with (a) ring system(s). Strychnine andits analogues are also useful phytochemicals that find use in medicinesand homeopathic treatments.

In another embodiment of the invention, it has been found that theso-called hTAS2R14 receptor is activated by tyrosine, in particularL-tyrosine, and other bitter tasting amino acids including leucine,histidine, phenylalanine and tryptophan.

In another embodiment of the invention, it has been found that theso-called hTAS2R38 receptor is activated by acetylthiourea,N,N-dimethylthioforminamide, N,N′-diphenylthiourea, N-ethylthiourea,2-imidazolidinethione, 4(6)-methyl-2-thiouracil, N-methylthiourea,phenylthio-carbamid, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil,tetramethylthiourea, thioacetamide, thioacetanilide, 2-thiobarbituricacid, and 2-thiouracil.

From these studies certain inferences can be drawn regarding theaffinity of the compounds, which activate the hTAS2R38 receptor. Thus,for the promotion of activation derivatives of 2-thiouracil according tofollowing formula are preferred compounds.

Whereas R in this formula can be hydrogen, it is preferred that R is asubstituent selected from C₁-C₁₀ alkyl, which may be branched, linear orcyclic as appropriate, particularly preferred alkyls are methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl oriso-pentyl residues; lower alkenyl residues, preferably having two,three, four or five carbon atoms; lower alkynyl residues, preferablyhaving two, three, four or five carbon atoms, which can in a preferredembodiment be further substituted with F, Cl, Br, NH₂, NO₂, OH, SH, NH,CN, aryl, heteroaryl, COH or COOH group; heteroaryl, e.g. benzofuran andcumarin; aryl, e.g. phenyl, naphtyl; F, Cl, Br, NH₂, NO₂, OH, SH, NH,CN, aryl, alkylaryl, heteroaryl, alkylheteroaryl, COH or COOH group. Ina further embodiment the carbon atom at the 4 position can substitutedwith —O—R¹, in which R₁ can have the same meaning as outlined above forR.

Another general structure of compounds having affinity for hTAS2R38 andwhich are thus suitable for activation of hTAS2R38 is depicted by thefollowing formula:

In this formula R₂, R₃, and R₄ can each independently of each other havethe meaning H; alkyl, in particular lower alkyl (C₁-C₅, e.g. methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-pentyl oriso-pentyl); substituted alkyl; alkenyl, in particular lower alkenyl(C₂-C₅); substituted alkenyl; alkinyl, in particular lower alkinyl(C₂-C₅); substituted alkinyl: alkanal, in particular lower alkanal (e.g.—COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃); aryl, in particular phenyl;substituted aryl; heteroaryl; substituted heteroaryl; alkylaryl, inparticular benzyl; substituted alkylaryl; alkylheteroaryl; substitutedalkylheteroaryl aminoalkyl, in particular —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂;substituted aminoalkyl; aminoketone, in particular —NHCOCH₃; substitutedaminoketone; aminoaryl, in particular —NH-Ph; substituted aminoaryl; CN;NH₂; Halogen, in particular F, Cl, and Br; NO₂. In a preferredembodiment R₂ or R₃ and R₄ can form a ring, preferably a four, five,six, seven or eight membered hetero cycle, which in a preferredembodiment is an aromatic hetero cycle. The residue of R₂ or R₃, whichis not involved in the formation of the ring structure can have any ofthe meanings as outlined above. In a further preferred embodiment atleast one of R₂ or R₃ has the meaning alkanal, preferably lower alkanalas outlined above. In case that only one of R₂ or R₃ has the meaningalkanal, than the other substituent preferably has the meaning H.

In a preferred embodiment R₂ is selected from the group consisting of H,CH₃ and Ph, R₃ is selected from the group of H, CH₃ and Ph and R₄ isselected from the group consisting of H, CH₃, NH—Ph, —NHCH₂CH₃,—NHCH₂CH₃, —NHCH₃, and —N(CH₃)₂.

In another embodiment of the invention, it has been found that theso-called hTAS2R43 receptor is activated by saccharin, derivativesthereof and other sulfoneimids.

In another embodiment of the invention, it has been found that theso-called hTAS2R44 receptor is activated by saccharin and acesulfame K,derivatives thereof and other sulfoneimids.

In another embodiment of the invention, it has been found that theso-called hTAS2R45, hTAS2R46 and hTAS2R48 receptor is activated byabsinthine derivatives thereof and other sulfoneimids.

The skilled person will appreciate that having regard to thestructure-function information provided by the present invention, it ispossible to compile libraries of molecules to find inhibitors of thebitter response of the disclosed hTAS2R in particular of the hTAS2R10,14, 16, 38, 43, 44, 45, 46, and 48, which are triggered by the aboveoutlined specific bitter substance(s). Such inhibitors, and librariescomprising same, form other aspects of the pre-sent invention. A stillfurther aspect of the invention relates to the use of such inhibitors infood or pharmaceutical compositions containing bitter tastants such asreferred to herein above, for the elimination or suppression of bittertaste perception.

In practicing the various aspects and embodiments of the presentinvention in relation to cloning receptors, elucidating ligand-receptorpairs, and finding modulators of the bitter response of receptors,recourse is made to conventional techniques in molecular biology,microbiology and recombinant technology. Accordingly, the skilled personis fully apprised of such techniques and as such they are hereaftertreated only summarily in order to more fully describe the context ofthe present invention.

In order to express cDNAs encoding the receptors, one typicallysubclones receptor cDNA into an expression vector that contains a strongpromoter to direct transcription, a transcription/translationterminator, and a ribosome-binding site for translational initiation.Suitable bacterial promoters are well known in the art, e.g., E. coli,Bacills sp., and Salmonella, and kits for such expression systems arecommercially available. Similarly eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The eukaryotic expression vector maybe, for example an adenoviral vector, an adeno-associated vector, or aretroviral vector.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the receptor-encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operatively linked to the nucleic acid sequence encoding thereceptor and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding the receptor may typically be linked to amembrane-targeting signal such as the N-terminal 45 amino acids of therat Somatostatin-3 receptor sequence to promote efficient cell-surfaceexpression of the recombinant receptor. Additional elements of thecassette may include, for example enhancers.

An expression cassette should also contain a transcription terminationregion downstream of the structural gene to provide for efficienttermination. The termination region may be obtained from the same geneas the promoter sequence or may be obtained from different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23D, and fusionexpression systems such as GST and LacZ, but there are many more knownin the art to the skilled person that can be usefully employed.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A.sup.+,pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, pcDNA3.1, pIRES and anyother vector allowing expression of proteins under the direction of theSV40 early promoter, SV40 late promoter, metallothionein promoter,murine mammary tumor virus promoter, Rous sarcoma virus promoter,polyhedrin promoter, or other promoters shown effective for expressionin eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding drugresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particular drugresistance gene chosen is not critical, any of the many drug resistancegenes known in the art are suitable. The prokaryotic sequences areoptionally chosen such that they do not interfere with the replicationof the DNA in eukaryotic cells, if necessary.

Standard transfection methods can be used to produce bacterial,mammalian, yeast or insect cell lines that express large quantities ofthe receptor, which are then purified using standard techniques.

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,liposomes, microinjection, plasma vectors, viral vectors and any of theother well known methods for introducing cloned genomic DNA, cDNA,synthetic DNA or other foreign genetic material into a host cell. It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the receptor.

After the expression vector is introduced into the cells, thetransfected cells may be cultured under conditions favoring expressionof the receptor, which is recovered from the culture using standardtechniques. For example the cells may be burst open either mechanicallyor by osmotic shock before being subject to precipitation andchromatography steps, the nature and sequence of which will depend onthe particular recombinant material to be recovered. Alternatively, therecombinant protein may be recovered from the culture medium in whichthe recombinant cells had been cultured.

The activity of any of the receptors described herein can be assessedusing a variety of in vitro and in vivo assays to determine functional,chemical, and physical effects, e.g., measuring ligand binding,secondary messengers (e.g., cAMP, cGMP, IP₃, DAG, or Ca²⁺) ion flux,phosphorylation levels, transcription levels, neurotransmitter levels,and the like. Furthermore, such assays can be used to test forinhibitors of the receptors as is well known in the art.

Samples or assays that are treated with a potential receptor inhibitormay be compared to control samples without the test compound, to examinethe extent of modulation. Control samples (untreated with inhibitors)are assigned a relative receptor activity value of 100. Inhibition ofreceptor activity is achieved when the receptor activity value relativeto the control is lower, and conversely receptor activity is enhancedwhen activity relative to the control is higher.

The effects of the test compounds upon the function of the receptors canbe measured by examining any of the parameters described above. Anysuitable physiological change that affects receptor activity can be usedto assess the influence of a test compound on the receptors of thisinvention. When the functional consequences are determined using intactcells or animals, one can measure a variety of effects such as changesin intracellular secondary messengers such as Ca²⁺, IP3 or cAMP.

Preferred assays for G-protein coupled receptors include cells that areloaded with ion sensitive dyes to report receptor activity. In assaysfor identifying modulatory compounds, changes in the level of ions inthe cytoplasm or membrane voltage will be monitored using an ionsensitive or membrane voltage fluorescent indicator, respectively. ForG-protein coupled receptors, promiscuous G-proteins such as G.alpha.15and G.alpha.16 and chimeric G-proteins can be used in the assay ofchoice (see, for example, Wilkie et al., Proc. Nat. Acad. Sci. USA 188,10049-10053 (1991)). Such promiscuous G-proteins allow coupling of awide range of receptors.

Receptor activation typically initiates subsequent intracellular events,e.g., increases in second messengers such as IP₃, which releasesintracellular stores of calcium ions. Activation of some G-proteincoupled receptors stimulates the formation of inositol triphosphate(1133) through phospholipase C-mediated hydrolysis ofphosphatidylinositol (Berridge & Irvine, Nature 312, 315-21 (1984)). IP₃in turn stimulates the release of intracellular calcium ion stores.Thus, a change in cytoplasmic calcium ion levels, or a change in secondmessenger levels such as IP₃ can be used to assess G-protein coupledreceptor function. Cells expressing such G-protein coupled receptors mayexhibit increased cytoplasmic calcium levels as a result of contributionfrom both intracellular stores and via activation of ion channels, inwhich case it may be desirable, although not necessary, to conduct suchassays in calcium-free buffer, optionally supplemented with a chelatingagent such as EGTA, to distinguish fluorescence response resulting fromcalcium release from internal stores.

In a preferred embodiment, receptor activity is measured by expressingthe receptor in a heterologous cell with a promiscuous G-protein, suchas G.alpha.15, 16, or a chimeric G-protein that links the receptor to aphospholipase C signal transduction pathway. Optionally the cell line isHEK-293, although other mammalian cells are also preferred such as CHOand COS cells. Modulation of taste transduction is assayed by measuringchanges in intracellular Ca²⁺ levels, which change in response tomodulation of the receptor signal transduction pathway viaadministration of a molecule that associates with the receptor. Changesin Ca²⁺ levels are optionally measured using fluorescent Ca²⁺ indicatordyes and fluorometric imaging.

The type of assay described above with respect to G-protein coupledbitter taste receptors can, however, also be employed for theidentification of binding compounds, in particular agonists orantagonists of any G-protein coupled signalling molecule, in particularG-protein coupled receptor. Therefore, another aspect of the presentinvention relates to a process for the identification of agonists orantagonists of G-protein coupled signalling molecules comprising thesteps of:

-   (1) contacting a cell comprising a promiscuous G-protein like, for    example, G.alpha.15, 16, or a chimeric G-protein, and a G-protein    coupled signalling molecule, in particular receptor, with a the    potential agonist or antagonists of the signalling molecule;-   (2) determining whether the potential agonist or antagonists    agonizes or antagonizes the activity of the signalling molecule.

The activity of the signalling molecule and the increase or decrease ofthat activity in response to the potential agonist or antagonist can bedetermined as outlined above with respect to the identification ofbitter receptor taste activity. The respectively indicated percentincreases or decreases of the activity, which arc required to qualify asantagonist or agonist do apply mutatis mutandis. Additionally the term“contacting” has the meaning as outlined above. Preferably thesignalling molecule and/or the promiscuous G-protein has been introducedinto the cell. The type of cell, which are preferred are those indicatedabove.

In yet another embodiment, the ligand-binding domains of the receptorscan be employed in vitro in soluble or solid-state reactions to assayfor ligand binding. Ligand binding in a receptor, or a domain of areceptor, can be tested in solution, in a bilayer membrane attached to asolid phase in a lipid monolayer or vesicles. Thereby, the binding of amodulator to the receptor, or domain, can be observed using changes inspectroscopic characteristics, e.g. fluorescence, absorbance orrefractive index; or hydrodynamic (e.g. shape), chromatographic, orsolubility properties, as is generally known in the art.

The compounds tested as modulators of the receptors can be any smallchemical compound, or a biological entity, such as a protein, sugar,nucleic acid or lipid. Typically, test compounds will be small chemicalmolecules. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention, although knowledgeof the ligand specificity of an individual receptor would enable theskilled person to make an intelligent selection of interestingcompounds. The assays may be designed to screen large chemical librariesby automating the assay steps and providing compounds from anyconvenient source to assays, which are typically run in parallel (e.g.,in microtiter formats on microtiter plates in robotic assays). Theskilled person will understand that there are many suppliers oflibraries of chemical compounds.

Assays may be run in high throughput screening methods that involveproviding a combinatorial chemical or peptide library containing a largenumber of potential therapeutic, or tastant compounds (that arepotential ligand compounds). Such libraries are then screened in one ormore assays, as described herein, to identify those library members(particular chemical species or subclasses) that display a desiredcharacteristic activity. The compounds thus identified can serve as leadcompounds to further develop modulators for final products, or canthemselves be used as actual modulators.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art and no more needs to be stated here.

In the high throughput assays of the invention, it is possible to screenup to several thousand different modulators or ligands in a single day.In particular, each well of a microtiter plate can be used to run aseparate assay against a selected potential modulator, or, ifconcentration or incubation time effects are to be observed, every 5-10wells can test a single modulator. Thus, a single standard microtiterplate can assay about 100 (e.g., 96) modulators. If 1536 well plates areused, then a single plate can easily assay from about 100 to about 1500different compounds. It is possible to assay several different platesper day, assay screens for up to about 6,000-20,000 different compoundsis possible using the integrated systems of the invention.

Lead compounds found by assay technology herein above described, ordevelopment compounds formed from such leads can be administereddirectly to a human subject to modulate bitter taste. Alternatively,such compounds can be formulated with other ingredients of preparationsto be taken orally, for example, foods, including animal food, andbeverages, pharmaceutical or nutraceutical or homeopathic preparations.

Therefore, another aspect of the invention is a process for theproduction of foodstuffs or any precursor material or additive employedin the production of foodstuffs comprising the steps of the abovedescribed processes for the identification of a compound binding tohTAS2R or an antagonist of hTAS2R and the subsequent step of admixingthe identified compound or antagonist with foodstuffs or any precursormaterial or additive employed in the production of foodstuffs.

Bitter taste is a particular problem when orally administeringpharmaceuticals, which often have an unpleasant bitter taste. Inparticular in elderly persons, children and chronically ill patientsthis taste can lead to a lack of compliance with a treatment regimen. Inaddition in veterinary applications the oral administration of bittertasting pharmaceuticals can be problematic. Therefore, a further aspectof the invention is a process for the production of a nutraceutical orpharmaceutical composition comprising the steps of the processes of acompound binding to hTAS2R or an antagonist of hTAS2R and the subsequentstep of formulating the compound or antagonist with an active agent in apharmaceutically acceptable form.

Consequently, a further aspect of the invention is a foodstuff, inparticular animal food, or any precursor material or additive employedin the production of foodstuffs comprising an antagonist/inhibitordescribed above, preferably an antibody directed against one of thehTAS2Rs described herein, the extracellular domain of one of the hTAS2Rsdescribed herein or an inhibiting RNA.

Also comprised is a nutraceutical or pharmaceutical compositioncomprising an antagonist/inhibitor as described above, preferably anantibody directed against one of the hTAS2Rs described herein, theextracellular domain of one of the hTAS2Rs described herein or aninhibiting RNA and an active agent, which preferably inhibits a bittertaste, and optionally a pharmaceutically acceptable carrier.

The amount of compound to be taken orally must be sufficient to effect abeneficial response in the human subject, and will be determined by theefficacy of the particular taste modulators and the existence, nature,and extent of any adverse side-effects that accompany the administrationof a particular compound. There now follows a series of examples thatserve to illustrate the invention, not to limit.

A further aspect of the present invention is the use of a polynucleotideas described above, a vector as described above, an antibody asdescribed above or an antagonist/inhibitor of as described above,preferably an antibody directed against one of the hTAS2Rs describedherein, the extracellular domain of one of the hTAS2Rs described hereinor an inhibiting RNA for the manufacture of a medicament for thetreatment of an abnormally increased or decreased sensitivity towards abitter substance.

Techniques associated with detection or regulation of genes are wellknown to skilled artisans. Such techniques can be used, for example, forbasic research on bitter receptors and to diagnose and/or treatdisorders associated with aberrant bitter receptor expression.

The following examples are merely illustrative of the present inventionand should not be construed to limit the scope of the invention asindicated by the appended claims in any way. The contents of the U.S.provisional application Ser. No. 60/413,298 the priority of which isclaimed is hereby incorporated by reference in its entirety.

EXAMPLE 1 Cloning of the hTAS2R Genes

Human genomic DNA was isolated from HEK293 cells using the E.Z.N.A.Blood DNA Kit II (Peqlab) and the various hTAS2Rs were amplified by PCRusing gene-specific primers that span the complete coding region of theindividual hTAS2R genes. Reaction parameters were: 4 cycles; 1 min, 94°C.; 1 min, 64° C.; 1.5 min 68° C. using Advantage 2 polymerase(Clontech). 5% of the reaction served then as template for furtheramplification with Pfu DNA polymerase (Promega): 30 cycles; 1 min, 94 C;1 min, 64° C.; 3 min. 72° C. The hTAS2R amplicons were then sub-clonedinto a cassette based on pcDNA5-FRT (Invitrogen). The cloning cassettecontains the first 45 amino acids of the rat somatostatin type 3receptor (as is further described by Meyerhof et al., Proc. Nat. Acad.Sci. USA, 89, 10267-10271 (1992)) as a cell surface-targeting signal atthe N-terminus. The C-terminus contained the herpes simplex virus (HSV)glycoprotein D epitope which does not interfere with signaling ofheptahelical receptors and can be used for immunocytochemistry using anantibody that binds specifically to the HSV glycoprotein D epitope (seeRoosterman et al, J. Neuroendocrinol, 9, 741-751 (1997)). Comparison ofthe DNA sequences of at least four clones identified mutations generatedduring PCR and this avoided picking mutated clones. We compared theamino acid sequences using the AlignX program of the Vector NTI™ Suite(InforMax).

Using the above-described method, DNA sequences encoding all 24 bitterreceptors identified by applicant were cloned. As indicated above, theywere derived by a PCR-based method using genomic DNA as the template.Since all of the 24 genomic sequences lack introns, the DNA clonesobtained had the same sequences as corresponding cDNA clones derived byreverse transcription-PCR(RT-PCR) of mRNA from cells expressing therelevant polypeptides would have.

EXAMPLE 2 Immunocytochemistry

Batches of HEK293 cells were separately transiently transfected withexpression vectors (pCDN5/FRT; Invitrogen) containing each of the 24above described coding sequences using lipofectamine 2000 (Invitrogen)and aliquots of the resulting cell populations were separately seeded onpolylysine-coated coverslips. At 24 h post transfection they were washedwith phosphate buffered saline (PBS), cooled on ice and added 20microgram/ml biotin-labeled concanavalin A (Sigma) for 1 h, which bindsto cell surface glycoproteins. Thereafter, the cells were fixed for 5min in methanol/acetone (1:1) and then permeabilized for 4 min with0.25% Triton X-100. In order to reduce nonspecific binding thecoverslips were incubated in 2% goat serum. Thereafter, anti-HSVglycoprotein D antiserum (Novagen, 1:10,000) was added to detect thechimeric receptors that, as described above, would have a HSVglycoprotein epitope fused to their C-termini, and Texas Red-Avidin D(Vector, 1:200) has added to stain the cell surface and incubationcontinued overnight at 4° C. Such C-termini are intracellular and forthis reason it is necessary to permeabilize the cells to permit entry ofthe HSV glycoprotein D epitope-specific antibody molecules into them.After washing (5× in PBS, RT) Alexa488-conjugated goat anti-mouseantiserum (Molecular Probes, 1:1000) was added and incubation continuedat room temperature for 1 h. Finally, the cells were embedded inFluorescent Mounting Medium (Dako) and analyzed using a Leica TCS SP2Laser Scan Inverted microscope. The preparations were scannedsequentially with an argon/krypton laser (488 nm) to excite the Alexa488dye and with a green-helium-neon laser (543 nm) to excite the Texas Reddye. The spectral detector recorded light emission at 510-560 nm and580-660 nm, respectively. Images of 1024×1024 pixels were processed withCorel PHOTO-PAINT 10.0 (Corel Corporation) and printed on a Tektronixcolor laser printer. The immunocytochemical data permitted calculationof the proportion of cells expressing recombinant receptors (greenfluorescent cells divided by total cell number in a microscopic field)and the proportion of cells that display expression of TAS2Rs at theplasma membrane level (number of cells with colocalization of green andred fluorescence divided by the number of green fluorescent cells). Ofthe 24 transfectant lines tested, all were found to express the encodedpolypeptides. The proportion of receptor-expressing cells in the varioustransfectant lines ranged from about 10% to about 35%.

EXAMPLE 3 Heterologous Expression of hTAS2R Receptors

A fluorescence imaging plate reader (FLIPR, Molecular Devices) was usedto functionally screen cell populations transiently transfected withexpression vectors encoding the above-described 24 bitter receptors andto establish concentration-response curves for hTAS2R16 and hTASR10. Thesingle-cell calcium imaging technique was also employed to demonstratereceptor selectivity and crossdesensitization. For the FLIPR experimentsthe HEK293/15 cells were grown to 50% confluence. The cells were thenseeded at a density of 3×10³ cells per well into 96-well black-wall,clear-bottom microtiter plates (Greiner). After 48 h the cells in eachwell were transfected using Lipofectamine 2000 and 24-30 h later wereloaded with FIuo4AM (Molecular Probes). Thereafter they were stimulatedwith bitter compounds (SigmaAldrich, further purified by reversed-phaseHPLC to >99% purity). Calcium signals were recorded simultaneously fromeach well at 1 Hz at 510 nm after excitation at 488 nm and therecordings were corrected for cell density. The responses of five wellscontaining cells expressing the same receptor and that received the samestimulus (i.e., the same compound at the same concentration) wereaveraged. Calcium traces were subtracted that were determined intriplicate of mock-transfected cells stimulated with the sameconcentration of tastant. The calculations rest on at least fourindependent transfection experiments. Plots of the amplitudes versusconcentrations fitted by nonlinear regression to the functionf(x)=100/(1+(EC₅₀/x)_(nH)), with x=agonist concentration and nH=Hillcoefficient permitted calculation of EC₅₀ values and threshold values ofactivation.

EC₅₀ and threshold values obtained with hTAS2R16-expressingtransfectants are shown in Table 1 below and the results are describedin Example 4.

In separate experiments, hTAS2R10-expressing transfectants were found tohave a threshold of activation of approximately 0.1 μM and a EC₅₀ of5-20 μM using strychnine as the test compound. Similar results wereobtained with brucine.

Single-cell Ca²⁺ imaging was performed with the hTAS2R16-transfectedHEK293/15 cells as described in Cell 95, 917-926 (1998), but with thefollowing modifications: The Till Photonics imaging system (Munich,Germany) was used in which a monochromator is connected by a quartzfiber lightguide and an epifluorescence condenser to an inverted OlympusIX50 microscope equipped with a UApo/340 40×1.35 oil-immersion lens. 30h post-transfection, FURA-2AM-loaded cells were sequentially illuminatedin 5 s intervals for 3-10 ms, first at 340 nm, then at 380 nm, onlineratioed light emissions at 510 nm (340/380) and monitored the images viaan intensified, cooled CCD camera. The 5 s interval camera pictures ofall cells in the microscope field of vision permanently were stored andanalyzed offline. 10-15% of all cells in the camera field responded toagonists in transient transfection experiments. The proportion ofresponders was about half of that found by immunocytochemistry, probablyreflecting a sub-optimal signal transduction. Responses were notobserved in mock-transfected cells. Isoproterenol (10 microMolar) wasused at the end of all experiments to stimulate endogenousbetaadrenergic receptors, proving a functional G_(alpha 15) dependentsignal transduction cascade.

For RT-PCR and in-situ hybridization work, human RNA (Clontech) waspurchased or it was isolated from surgical tongue specimens with peqGOLDRNAPure (Peqlab) and the preparations digested with DNase I(Invitrogen). Following cDNA synthesis (Smart cDNA synthesis Kit,Clontech) hTAS2R16 cDNA was PCR-amplified (39 cycles, 1 min 94° C., 1min 64° C., 1 min 72° C.) using specific forward and reverse primerswith overhangs containing EcoRI or NotI sites SEQ ID Nos 51 and 52 andthe amplicons analyzed on agarose gels. Subcloning and sequencingdemonstrated the identity of the amplified bands. Approximately 15micrometer cryo-sections of human tongue specimens containing vallatepapilla at 65° C. were processed and hybridized with a hTAS2R16riboprobe spanning the complete coding region and generated fromhTAS2R16 cDNA. The in-situ hybridization method used was essentially thesame as that described in Nature, 413, 631-635 (2001) except that theriboprobe was conjugated with biotin and an alkaline phosphatase-avidinconjugate was used for detection. This experiment indicated that TAS2R16mRNA is expressed in vallate papilla which are known to perceive bittertaste.

EXAMPLE 4 Human Taste Experiments

15 experienced panelists in a sensory panel room at 22-25° C. determinedbitter thresholds on three different sessions using a triangle test withtap water as solvent, according to methodology set out in J. Agric. FoodChem., 49, 231-238 (2001), or Mailgaard M et al, “Sensory EvaluationTechniques” (CRC Press LLC, New York 1999). For dose-response relations,bitter tastant concentration series were presented to 10 trainedpanelists in random order. The panelists ranked the samples inincreasing order of intensity and, for each concentration, evaluatedbitterness intensity on a scale from 0 to 5 (ref. 24). The dose-responsecurves of three different sessions were averaged. The intensity valuesbetween individuals and separate sessions differed by not more than 0.5units.

To investigate adaptation, the 8 panelists first maintained aqueoussolutions (5 ml) of phenyl-β-D-glucopyranoside (8 mM),phenyl-alpha-D-glucopyranoside (180 mM), salicin (8 mM), or helicin (8mM) for 15 s in their oral cavities and evaluated the bitter intensityas described above. After 30 min, they kept a denatonium benzoatesolution (5 ml, 0.0003 mM) for 15 s in their mouth and evaluated itsbitterness. The panelists spat off the denatonium benzoate solution,took up the phenyl-β-D-glucopyranoside or thephenyl-alpha-Dglticopyranoside solutions orally for 120 s or 180 s andjudged their bitterness intensity after 15, 30, 60, 120 and 180 s.Thereafter, the panelists spat off these solutions and then sequentiallytook up salicin, helicin (5 ml, 8 mM) and denatonium benzoate (5 ml,0.0003 mM) and evaluated bitterness intensities of these solutions after15 s. After an additional 30 min, the first experiment was repeated. Thedata of three different sessions for each panelist were averaged.Intensity values between individuals and separate sessions differed bynot more than ±0.5 units.

Results of in vitro assays (FLIPR) and human taste experiments are shownin Table 1 below.

TABLE II Com- Threshold Value (mM) EC₅₀ (mM) pound FLIPR Human FLIPRHuman 1 0.07 +/− 0.02  0.1 +/− 0.05 1.1 +/− 0.1 0.7 +/− 0.2 2 0.07 +/−0.02 0.2 +/− 0.1 1.4 +/− 0.2 1.1 +/− 0.3 3 0.3 +/− 0.1 0.4 +/− 0.1 2.3+/− 0.4 2.2 +/− 0.7 4 0.5 +/− 0.2 0.9 +/− 0.3 5.8 +/− 0.9 5.4 +/− 1.8 51.5 +/− 0.5 n.d. n.d. n.d. 6 0.4 +/− 0.1 0.2 +/− 0.1 1.0 +/− 0.1 1.4 +/−0.4 7 15 +/− 6  32 +/− 11 n.d. 320 +/− 108 8 2.3 +/− 0.9 n.d. 20 +/− 3.4n.d. 9 4 +/− 2 4 +/− 1 n.d. n.d. 10 n.r. 40 +/− 13 n.r. n.d. 11 n.r. 9+/− 3 n.r. 50 +/− 17 1 = phenyl-beta-D-glucopyranoside; 2 = salicin; 3 =helicin; 4 = arbutin; 5 = 2-nitro-phenyl-beta-D-glucopyranoside; 6 =naphthyl-beta-D-pyranoside; 7 = methyl-beta-D-lucopyranoside; 8 =amygdalin; 9 = esculin; 10 = phenyl-beta-D-galactopyranoside; 11 =phenyl-alpha-D-glucopyranoside. n.d. = not data due to solubilityproblems or toxicity or artifacts in vitro. n.r. = No response up to 100mM.

The FLIPR results provide the threshold concentration of the compounds(nM) at which point the receptor detects the compounds. The EC₅₀ resultsexpress the concentration of the compound wherein the receptor signal isat 50%, and is a representation of the affinity of a receptor for acompound.

The results show that the in vitro FLIPR measurements for salicinclosely resemble the human taste study results. This bitter-tastingcompound has known anti-pyretic and analgesic action, and the resultssuggest that in vitro assays using hTAS2R16 may represent a useful toolto find compounds that suppress or eliminate the bitter response to thiscompound. Also, for all the other tested beta-glucopyranosides, theclose correspondence of Threshold Concentration and EC₅₀ results suggestthat hTAS2R16 is a cognate human receptor for these class of bittercompounds. In contrast, the related structures (see compounds 10 and 11)show 90- to 400-fold higher Threshold Concentrations, which indicatesthat this receptor is rather selective, and that these bitter compoundsactivate different receptors.

Adaptation frequently occurs in sensory systems and means that stimulielicit reduced responses upon prolonged or repeated stimuluspresentations. Repeated stimulation of hTAS2R16-expressing cells withphenyl-beta-D-glucopyranoside resulted in largely diminished responsesto salicin as well. This cross-desensitization occurred among the othertested beta-pyranosides and was fully reversible. It resembleshomologous desensitization of agonist-occupied heptahelical receptorsmediated by GRKs, i.e. specific kinases, and arresting. We also observedadaptation in the human test panel that initially scoredphenyl-beta-D-glucopyranoside, salicin and helicin as equally intenselybitter. The bitterness of phenyl-beta-D glucopyranoside declined duringprolonged stimulation and the test panel perceived salicin and helicinalso as less bitter, but not the unrelated bitter substance denatoniumbenzoate, which cannot activate TAS2R16. Adaptation was fullyreversible. On the opposite, the phenyl-alpha-D-glucopyranoside failedto cross-adapt with all tested beta-D-glucopyranosides, although its ownbitter response desensitized strongly. This indicates thatbeta-glucopyranosides signal through a common mechanism most likelyinvolving hTAS2R16 as a bitter taste receptor while the alpha-isomeractivates a separate receptor. A recent human psychophysical study alsorevealed cross-adaptation amongst two bitter amino acids but not betweenthe two bitter amino acids and urea, suggesting the existence ofdistinct receptors for the bitter amino acids and urea. Although most,if not all, bitter receptors are present in the same subset of tastereceptor cells, adaptation to specific bitter stimuli can be explainedif bitter receptors were subject to homologous desensitization.

EXAMPLE 5 Heterologous Expression of hTAS2R

Transient transfection of TAS2Rs into HEK-293T-Gα16gustdicin44 cells. Wecloned the DNAs of all human putative bitter responsive receptors intopcDNA5/FRT (Invitrogen) by PCR-methods and transiently transfected theplasmids with lipofectamine 2000 (Invitrogen) intoHEK-293T-Gα16gustducin44 cells grown to 50% confluence. These cellsstably express a chimeric G protein constructed from human Gα16 and ratgustducin. Finally, we seeded the transfected cells at a density of3×10³ cells per well into 96-well black-wall, clear-bottom microtiterplates (Greiner).

Co-transfection of TAS2Rs with gustducin and phospholipase-Cβ2 intoHEK-293 cells. Alternatively, we transfected simultaneously plasmid DNAsencoding one of the TAS2Rs, phosplolipase-Cβ2 and α-gustducin intoHEK-293 cells using the lipofectamine method. Additional cotransfectionof G-protein β and γ-subunits may improve the bitter tastant-inducedresponses. Thereafter, the transfected cells were seeded at a density of3×10³ cells per well into 96-well black-wall, clear-bottom microtiterplates (Greiner).

Fluorometric Imaging Plate Reader (FLIPR) assay 24-30 h later, the cellswere loaded with 4 μM FLUO-4/AM (Molecular Probes) and 0.04% PluronicF-127 (Molecular Probes) in Hepes-buffered saline (HBS), 140 mM NaCl, 5mM KCl, 2.5 mM CaCl₂, 10 mM Hepes, 10 mM glucose and 2.5 mM probenicide,pH 7.4, for 1 hour at 37° C. Thereafter, cells were gently washed in HBSby an automated plate washer (Denley Cellwash, Labsystems) andtransferred to the FLIPR (Molecular Devices). The FLIPR integrates anargon laser excitation source, a 96-well pipettor, and a detectionsystem utilizing a Charged Coupled Device imaging camera. Fluorescenceemissions from the 96 wells were monitored at an emission wavelength of510 nm, after excitation with 488 nm (F488). Fluorescence data werecollected 1 min before and 10 min after stimulation. Data were collectedevery 6 s before and every 1 s after agonist stimulation. 50 μl of 3×concentrated agonists were delivered within 2 s by the integrated96-well pipettor to the wells containing 100 μl HBS. Agonist responseswere quantified using the amplitudes of the fluorescence peaks. Weaveraged the responses of five wells containing cells expressing thesame receptor and that received the same stimulus. Calcium traces weredetermined in triplicate of mock-transfected cells stimulated with thesame concentration of tastant. EC₅₀ values and plots of the amplitudesversus concentrations were derived from fitting the data by nonlinearregression to the function f(x)=100/[1+(EC₅₀/x)^(nH)], where x is theagonist concentration and nH is the Hill coefficient. The results forhTAS2R10 (Table II), hTAS2R14 (Table III), hTAS2R16 (Table IV), hTAS2R38(Table V), hTAS2R43 (Table VI), hTAS2R44 (Table VII), hTAS2R45 (TableVIII), hTAS2R46 (Table IX) and hTAS2R (Table X) are shown below.

TABLE III Identified agonists of hTAS2R10 Approx. thresh- SubstanceStructure old [mM] EC₅₀ [mM] Strychnine*

0.003 0.04 Brucine

0.01 0.06 Denatonium benzoate

0.003 0.07 Absinthine

0.01

TABLE IV Identified agonists of hTAS2R14 Substance Structure Reacts atL-Tyrosine

1 mM

TABLE V Identified agonists of hTAS2R16 Substance Structure Threshold[mM] EC₅₀ [mM] Naphtyl-β-D-Glucoside

0.4 ± 0.1 1.0 ± 0.1 Phenyl-β-D-Glucoside

0.07 ± 0.02 1.1 ± 0.1 Salicin

0.07 ± 0.02 1.4 ± 0.2 Helicin

0.3 ± 0.1 2.3 ± 0.4 Arbutin

0.5 ± 0.2 5.8 ± 0.9 2-Nitophenyl-β-D-Glucoside

0.3-1   Not determined 4-Nitrophenyl-β-D-Glucoside

1-3 Not determined Methyl-β-D-Glucoside*

15 ± 6  32 ± 11 Esculin

4-2 Not determined 4-Nitrophenyl-β-D-Thioglucoside

1-5 Not determined 4-Nitrophenyl-β-D-Mannoside

1-3 Not determined Amygdalin

2.3 ± 0.9  20 ± 3.4

TABLE VI Identified agonists of hTAS2R38 Approx. Threshold EC₅₀Substance Structure [μM] [μM] Acetylthiourea

2 15 N,N-Dimethyl-thioformamide

10 55 N,N′-Diphenylthiourea

0.3 2.3 N-Ethylthiourea

30 260 2-Imidazolidinethione(=N,N′-Ethylenethiourea)

10 not determined 4(6)-Methyl-2-thiouracil

20 180 N-Methylthiourea

100 estimated600-800 Phenylthiocarbamid(PTC)

0.3 2 6-Phenyl-2-thiouracil

0.15 0.5 6-Propyl-2-thiouracil(PROP)

0.3 2 Tetramethylthiourea

10-30 100 Thioacetamide

100 not determined Thioacetanilide

3 18 2-Thiobarbituric acid

reacts at 10 mM 2-Thiouracil

300 estimated2000

TABLE VII Identified agonists of hTAS2R43 Approx. Thres- SubstanceStructure hold [mM] EC₅₀ [mM] Saccharin

0.2 1.1 AcesulfameK

No response up to 10 mM

TABLE VIII Identified agonists of hTAS2R44 Approx. Thres- SubstanceStructure hold [mM] EC₅₀ [mM] Saccharin

0.2 estimated2-5 Acesulfame K

0.5 3

TABLE IX Identified agonists of hTAS2R45 Approx. Thres- SubstanceStructure hold [mM] EC₅₀ [mM] Absinthine

0.003 Not determined

TABLE X Identified agonists of hTAS2R46 Approx. Thresh- SubstanceStructure old [mM] EC₅₀ [mM] Absinthine

0.001 Not determined

TABLE XI Identified agonists of hTAS2R48 Approx. Thresh- SubstanceStructure old [mM] EC₅₀ [mM] Absinthine

0.03 Not determined

1. A process for identifying an antagonist of the bitter taste receptoractivity of the polypeptide encoded by a polynucleotide selected fromthe group consisting of: (a) a polynucleotide encoding at least a matureform of a polypeptide having the deduced amino acid sequence as shown inSEQ ID NO: 1; (b) a polynucleotide having the coding sequence, as shownin SEQ ID NO: 2 encoding at least a mature form of the polypeptidehaving the deduced amino acid sequence as shown in SEQ ID NO:1; (c) apolynucleotide encoding a derivative of a polypeptide encoded by apolynucleotide of any one of (a) to (b), wherein in said derivative oneto twenty amino acid residues are conservatively substituted compared tosaid polypeptide, and said derivative has bitter taste receptor activitywhen contacted with an agonist selected from the group consisting ofacetylthiourea, N,N-dimethylthioformamide, N,N′-diphenylthiourea,N-ethylthiourea, 2-imidazolidinethione, 4)6)-methyl-2-thiouracil,N-methylthiourea, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil,tetramethylthiourea, thioacetamide, thioacetanilide, 2-thiobarbituricacid and 2-thiouracil; (d) a polynucleotide which is at least 85%identical to a polynucleotide as defined in any one of (a) to (c) andwhich encodes a polypeptide having bitter taste receptor activity whencontacted with an agonist selected from the group consisting ofacetylthiourea, N,N-dimethylthioformamide, N,N′-diphenylthiourea,N-ethylthiourea, 2-imidazolidinethione, 4(6)-methyl-2-thiouracil,N-methylthiourea, 6-phenyl-2-thiouracil, 6-propyl-2-thiouracil,tetramethylthiourea, thioacetamide, thioacetanilide, 2-thiobarbituricacid and 2-thiouracil; and (e) a polynucleotide the complementary strandof which hybridizes under high stringency hybridization conditions to apolynucleotide as defined in any one of (a) to (d) and which encodes apolypeptide having bitter taste receptor activity when contacted with anagonist selected from the group consisting of acetylthiourea,N,N-dimethylthioformamide, N,N′-diphenylthiourea, N-ethylthiourea,2-imidazolidinethione, 4(6)-methyl-2-thiouracil, N-methylthiourea,6-phenyl-2-thiouracil, 6-propyl-2-thiouracil, tetramethylthiourea,thioacetamide, thioacetanilide, 2-thiobarbituric acid and 2-thiouracil;wherein said process comprises the steps of: (1) contacting saidpolypeptide, or a host cell genetically engineered with saidpolynucleotide or with a vector containing said polynucleotide, with anagonist of bitter taste receptor activity selected from the groupconsisting of acetylthiourea, N,N-dimethylthioformamide,N,N′-diphenylthiourea, N-ethylthiourea, 2-imidazolidinethione,4(6)-methyl-2-thiouracil, N-methylthiourea, 6-phenyl-2-thiouracil,6-propyl-2-thiouracil, tetramethylthiourea, thioacetamide,thioacetanilide, 2-thiobarbituric acid, 2-thiouracil and functionalderivatives thereof; (2) contacting said polypeptide, or a host cellgenetically engineered with said polynucleotide or with a vectorcontaining said polynucleotide, with a potential antagonist; and (3)determining whether the potential antagonist antagonizes the bittertaste receptor activity of said polypeptide.
 2. The process of claim 1wherein steps (1) and (2) are carried out concomitantly.
 3. The processof claim 1 wherein step (2) is carried out prior to step (1).
 4. Aprocess selected from the group consisting of: A. a process for theproduction of a food or any precursor material or additive employed inthe production of foodstuffs comprising the steps of: (1) identifying anantagonist according to the process of claim 1; and (2) admixing theidentified antagonist with a foodstuff precursor material or additiveemployed in the production of foodstuffs; and B. a process for theproduction of a nutraceutical or pharmaceutical composition comprisingthe steps of; (1) identifying an antagonist according to the process ofclaim 1; and (2) formulating the identified antagonist with an activeagent in a pharmaceutically acceptable form.